A Tandem Oligonucleotide Approach for SNP-Selective RNA Degradation Using Modified Antisense Oligonucleotides

Abstract

Antisense oligonucleotides have been studied for many years as a tool for gene silencing. One of the most difficult cases of selective RNA silencing involves the alleles of single nucleotide polymorphisms, in which the allele sequence is differentiated by a single nucleotide. A new approach to improve the performance of allele selectivity for antisense oligonucleotides is proposed. It is based on the simultaneous application of two oligonucleotides. One is complementary to the mutated form of the targeted RNA and is able to activate RNase H to cleave the RNA. The other oligonucleotide, which is complementary to the wild type allele of the targeted RNA, is able to inhibit RNase H cleavage. Five types of SNPs, C/G, G/C, G/A, A/G, and C/U, were analyzed within the sequence context of genes associated with neurodegenerative disorders such as Alzheimer’s disease, Parkinson’s disease, ALS (Amyotrophic Lateral Sclerosis), and Machado-Joseph disease. For most analyzed cases, the application of the tandem approach increased allele-selective RNA degradation 1.5–15 fold relative to the use of a single antisense oligonucleotide. The presented study proves that differentiation between single substitution is highly dependent on the nature of the SNP and surrounding nucleotides. These variables are crucial for determining the proper length of the inhibitor antisense oligonucleotide. In the tandem approach, the comparison of thermodynamic stability of the favorable duplexes WT RNA-inhibitor and Mut RNA-gapmer with the other possible duplexes allows for the evaluation of chances for the allele-selective degradation of RNA. A larger difference in thermodynamic stability between favorable duplexes and those that could possibly form, usually results in the better allele selectivity of RNA degradation.

Modified RNA triplexes: Thermodynamics, structure and biological potential

“…These observations reflect the incorporation of modified nucleotides into the TFO strand. In addition, the number and position of LNA and 2-thioU nucleotides affect the biological potential of TFOs 48 . Despite the lack of direct correlation between the binding affinities of TFOs and their GFP gene silencing, simultaneous incorporation of the two modifications (LNA and 2-thioU) into TFO strand is beneficial for triplex formation.…”

Influence of mismatched and bulged nucleotides on SNP-preferential RNase H cleavage of RNA-antisense gapmer heteroduplexes

“…Given the RNA-antisense oligonucleotide interactions, in this SNP, a destabilizing A-dC single mismatch occurs in the duplex of the WT RNA-mutant-complementary gapmer eKM. Due to the mismatch, binding of the eKM gapmer to WT RNA 693 (A allele) is about two times weaker than to the complementary mutant RNA (G allele) 26 . However, the central position of this mismatch type did not influence RNase HI selectivity.…”

“…Targeting SNPs by ASOs to distinguish between wild type and mutant alleles is based on the occurrence of a single mismatch in one of the two RNA/ASO duplexes. Differentiation between the cleavage rates of these duplexes by RNase H might vary depending on the mismatch type and position with the ASO/target RNA duplex 26 . Single mismatch discrimination cleavage of a target RNA with RNase H was reported by Giles et al ., who used chimeric methylphosphonodiester/phosphodiester oligonucleotides 27 .…”

Machado–Joseph disease/spinocerebellar ataxia type 3: lessons from disease pathogenesis and clues into therapy

“…,b; Magner et al . ). In 2013, Evers and colleagues showed that an oligonucleotide‐mediated exon skipping strategy targeting the polyQ repeat did not impact ataxin‐3 molecular function, and the new truncated protein did not display any toxicity in vivo (Evers et al .…”